Adult murine intestine epithelial cell chromatin accessibility
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA939158 | ENA |
REPOSITORIES: ENA
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Project description:Microarray analyses of laser microdissected murine intestinal epithelial cells under the control of maternal inflammation at 17.5 days post conception demonstrates that maternal inflammation differentially regulates 2174 (iWT) and 3345 (iARE) genes in fetal tissue, but these transcriptional profiles were overwritten in the postnatal environment dominated by tissue inflammation at 8 weeks postnatal. We isolated 50 ng total RNA out of laser microdissected fetal and adult intestinal epithelial cells from the distal ileum and performed microarrays to address whether maternal inflammation programs the fetal intestine towards TNF-driven pathology Conventionally raised heterozygous TnfM-NM-^TARE/+ and Tnf+/+ wild type mice on C57BL/6 background were used for this study. Female Tnf+/+ wild type (WT) mice were bred with male TnfM-NM-^TARE/+ (ARE) mice and vice versa, generating offspring from healthy WT dams (WT and ARE) and inflamed ARE dams (iWT and iARE). On day 17.5 post conception (dpc) and 8 weeks postnatal the fetuses and offspring were sacrificed.The gut was freshly frozen in Optimal Cutting Temperature (O.C.T.) embedding compound (Sakura Finetek, Torrance, USA) on dry ice and stored at -80M-BM-0C until laser microdissection.
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Project description:Despite the crucial function of the small intestine for nutrient uptake our understanding of the molecular events underlying the digestive function is very rudimentary. For example, it has only recently become evident that not all absorbed triacylglycerol (TAG) is immediately secreted in the form of chylomicrons (CM) by enterocytes. Instead, especially after high-fat challenges, parts of the re-synthesized TAG can be packaged into cytosolic lipid droplets (CLD) for transient storage in the endothelial layer of the small intestine. The reasons for this intermediate storage of TAGs are currently not completely understood. Several mechanisms, including alleviating lipotoxicity to enterocytes, limits in the rate of CM assembly or smoothening of the post-prandial peaks of blood hypertriglyceridemia have been suggested to explain this intermediate storage of TAG in CLD. Interestingly, rather than being evenly distributed across the small intestine, the proximal jejunum exhibits the highest TAG storage and CM secretion. Once stored in CLD, lipids are either remobilized in the inter-prandial phase or by various stimuli, e.g. a sequential meal containing either lipids or glucose. After remobilization, a triglyceride peak can be observed in the form of plasma CM well before lipids from the second meal have been digested in the intestinal lumen. To synthesize CM with lipids from CLD, CLD TAG have to be hydrolyzed to be transported across the ER membrane which can either be achieved by cytoplasmic TAG lipolysis or lipophagy. We hypothesize that correlating enzymatic activities of hydrolases with the reported distribution of TAG storage and chylomicron secretion in different sections of the small intestine is a promising strategy to pinpoint the key players in TAG remobilization. To rank hydrolases based on their relative activity in the different sections of the small intestine we have harnessed a serine hydrolase specific activity-based labeling strategy to label active hydrolases in freshly harvested enterocytes. Enrichment of activity labeled enzymes over control samples and abundance of enriched hydrolases from individual small intestine sections was analyzed using quantitative proteomics, resulting in a ranking of the most active hydrolases in 11 fractions of murine small intestine. Moreover, several clusters of enzymes showing similar activity distribution along the small intestine were identified.
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